The agricultural industry seeks more effective methods of crop protection. Increasingly, attention has turned to genetic manipulation of plant proteins to create safe and more effective crop protection chemicals and herbicide-resistant plants. One area of interest is the monomeric guanine nucleotide-binding proteins of the Ras superfamily. These proteins function in a variety of cellular processes including signaling, growth, immunity and protein transport (Barbacid et al., Annu. Rev. Biochem. 56:779-828 (1987); Bourne, Cell 53:669-671 (1988); Bourne et al., Nature, London 349:117-127 (1991); Gabig et al., J. Biol. Chem. 262:1685-1690 (1987); Goud et al., Nature, London 345:553-556 (1990); Hall, Science 249:635-640 (1990); Knaus et al., Science 254:1512-1515 (1991)). Adenosine diphosphate (ADP) ribosylation factors (ARFs) constitute one family of proteins of the Ras superfamily.
ARFs are 20-kDa GTP binding proteins and were initially identified as activators required for the cholera toxin-catalyzed ADP ribosylation of G.sub.S.alpha., the stimulatory guanine nucleotide-binding (G) protein of the adenylyl cyclase system (Schleifer et al., J. Biol. Chem. 257:20-23 (1982); Kahn et al., J. Biol. Chem. 259:6228-6234 (1984); Serventi et al., Current Topics in Microbiology and Immunology 175, pp. 43-67, Springer-Verlag, Berlin Heidelberg (1992)). In the presence of guanosine 5'-triphosphate (GTP) or a nonhydrolyzable GTP analogue, ARF serves as an allosteric activator of cholera toxin ADP-ribosyltransferase activity. ARFs have also been shown to stimulate the ADP-ribosylation of proteins unrelated to the adenylate cyclase system, simple guanidino compounds such as arginine and agmatine, as well as the auto-ADP-ribosylation of the choleragen A.sub.1 peptide (Noda et al., Biochim. Biophys. Acta 1034:195-199 (1990); Tsai et al., J. Biol. Chem. 263:1768-1772 (1988); Tsai et al., Proc. Natl, Acad. Sci., USA 84:5139-5142 (1987)).
ARFs are evolutionarily well-conserved and present in all eukaryotes from Giardia to mammals (Kahn et al., J. Biol. Chem. 263:8282-8287 (1988); Murtagh et al., J. Biol. Chem. 267:9654-9662 (1992); Tsai et al., J. Biol. Chem. 266:8213-8219 (1991); Tsuchiya et al., Biochemistry 28:9668-9673 (1989); Tsuchiya et al., J. Biol. Chem. 266:2772-2777 (1991)). Thus, ARFs are believed to be critical components of vesicular trafficking pathways in eukaryotic cells (Sollner et al., Cell Struct. Funct. 21:407-412 (1996)). ARF's ability to activate phospholipase D may mediate this process. (Brown et al., Cell 75:1137-1134 (1993)). It has been proposed that following its activation by ARF, phospholipase D hydrolyzes phosphotidylcholine. The phosphatidic acid produced by this reaction facilitates formation of stable binding sites for coatomer, leading to budding of coated vesicles (Ktistakis et al., J. Cell Biol. 134:295-306 (1996)).
ARFs have been localized to the Golgi apparatus of several types of cells by immunocytochemistry (Stearns et al., Proc. Natl. Acad. Sci., USA 87:1238-1242 (1990)). ARFs are required for association of nonclathrin coat proteins with intracellular transport vesicles (Serafini et al., Cell 67:239-253 (1991)) and also appear to be critical during an early step in endocytosis as well as in nuclear vesicle fusion (Boman et al., Nature, London 358:512-514 (1992); Lenhard et al., J. Biol. Chem. 267:13047-13052 (1992)). GTP binding and hydrolysis may be involved in binding of ARF to membranes. Furthermore, the nonhydrolyzable GTP analogue guanosine 5'-[.gamma.-thio]triphosphate (GTP.sub..gamma.S), but not GDP or ATP, promotes the association of cytosolic ARF with Golgi (Regazzi et al., Biochem. J. 275:639-644 (1991) or phospholipid membranes (Kahn et al., J. Biol. Chem. 266:15595-15597 (1991); Walker et al., J. Biol. Chem. 267:3230-3235 (1992)).
ARFs are active in their GTP-bound forms and inactive when GDP is bound. ARFs exhibit no detectable GTPase activity. The ratio of GTP/GDP that is bound is regulated by guanine nucleotide-exchange proteins and GTPase-activating proteins (Vitale et al., J. Biol. Chem. 272:3897-3904 (1997)). Additionally, myristoylation of the protein seems to be necessary for ARF function. However, it is not clear mechanistically how myristoylation affects ARF's function although it has been suggested to facilitate nucleotide exchange and enhance phospholipid-dependent stabilization of the GTP bound form (Franco et al., J. Biol. Chem. 270:1337-1341 (1995)). Some lipids and/or detergents, e.g., SDS, cardiolipin, dimyristoylphosphatidylcholine (DMPC)/cholate, enhance ARF activities (Bobak et al., Biochemistry 29:855-861 (1990); Noda et al., Biochim. Biophys. Acta 1034:195-199 (1990); Tsai et al., J. Biol. Chem. 263:1768-1772 (1988)).
Regardless of their mechanism of action, ARFs are thought to be essential for eukaryotic cell viability. Saccharomyces cerevisiae contains three genes encoding ARF proteins and the mutant containing the deletion of arf1/arf2 is not viable (Stearns et al., Mol. Cell. Biol. 10:6690-6699 (1990); Lee et al., J. Biol. Chem. 269:20931-20937 (1994)).
By molecular cloning from cDNA and genomic libraries, and PCR amplification of RNA transcripts, six mammalian ARFs, three yeast ARFs, and two Giardia ARFs have been identified (Bobak et al., Proc. Natl. Acad. Sci., USA 86:6101-6105 (1989); Monaco et al., Proc. Natl. Acad. Sci., USA 87:2206-2210 (1990); Murtagh et al., J. Biol. Chem. 267:9654-9662 (1992); Price et al., Proc. Natl. Acad. Sci., USA 85:5488-5491 (1988); Sewell et al., Proc. Natl. Acad. Sci., USA 85:4620-4624 (1988); Stearns et al., Mol. Cell. Biol. 10:6690-6699 (1990); Tsuchiya et al., J. Biol. Chem. 266:2772-2777 (1991)). Mammalian ARFs fall into three classes based on deduced amino acid sequences, gene structure, phylogenetic analysis and size (Lee et al., J. Biol. Chem. 267:9028-9034 (1992); Tsuchiya et al., J. Biol. Chem. 266:2772-2777 (1991)). Class I ARFs are ARFs 1-3; class II includes ARFs 4 and 5; and class III has ARFs 6. The high degree of conservation between cognate ARFs is evidence of evolutionary pressure to maintain individual identities (Price et al., Mol. Cell Biochem. 159:15-23 (1996).
Members of the ARF multigene family, when expressed as recombinant proteins in E. coli, display different phospholipid and detergent requirements (Price et al., J. Biol. Chem. 267:17766-17772 (1992)). Following synthesis in E. coli all of these ARFs had enhanced cholera toxin ADP-ribosyltransferase activity in the presence of GTP (Kahn et al., J. Biol. Chem. 266:2606-2614 (1991); Price et al., J. Biol. Chem. 267:17766-17772 (1992); Weiss et al., J. Biol. Chem. 264:21066-21072 (1989)).
In general, differences in the various mammalian ARF sequences are concentrated in the extreme amino and carboxyl portions of the proteins. Only three of seventeen amino acids including Met.sub.1 and Gly.sub.2, in the amino termini are identical among ARFs, and four amino acids in this region of ARFs 1-5 are missing in ARF 6 (Tsuchiya et al., J. Biol. Chem. 266:2772-2777 (1991)). Kahn et al. reported J. Biol. Chem. 267:13039-13046 (1992)) that the amino-terminal regions of ARF proteins form an .alpha.-helix and that this domain is required for membrane targeting, interaction with lipid, and ARF activity.
Even less is understood about the role of ARFs in plants, although cDNAs have been cloned from a number of plants including Arabidopsis (Regad et al., FEBS Letters 316:133-136 (1993); Lebas et al., Plant Physiol. 106:809-810 (1994)), rice (Higo et al., Plant Science 100:41-49 (1994)), potato (Szopa et al., Plant Cell Reports 14:180-183 (1994)), maize (Verwoert et al., Plant Mol. Biol. 27:629-633 (1995)), carrot (Kiyosue et al., Plant and Cell Physiology 36:849-856 (1995)), and barley (EP 681028). Antisense experiments in potato show an increase in the levels of a 40-kDa ribosylated protein and concomitant decrease in a 42-kDa protein although the function and identity of these proteins are not known (Szopa et al., Plant Physiol. 145:383-386 (1995)). Furthermore, no obvious morphological changes were observed in the antisense plants.
A need exists for improved crop protection methods. A method that has broad applicability for screening for candidate compounds and the production of transgenic plants would have great commercial value. No such method involving nucleic acid fragments encoding soybean adenosine diphosphate (ADP) ribosylation factors (ARF's) was previously known.